CN110563849A

CN110563849A - anti-VEGF-anti-PD 1 bispecific antibody with brand new sequence

Info

Publication number: CN110563849A
Application number: CN201910734481.1A
Authority: CN
Inventors: 汪国兴; 程联胜; 胡思怡; 袁红; 武婷; 樊丽
Original assignee: ANHUI RUBIOX-VISION BIOTECHNOLOGY Co Ltd
Current assignee: ANHUI RUBIOX-VISION BIOTECHNOLOGY Co Ltd
Priority date: 2019-08-09
Filing date: 2019-08-09
Publication date: 2019-12-13
Anticipated expiration: 2039-08-09
Also published as: CN110563849B

Abstract

The invention relates to an anti-VEGF-anti-PD-1 bispecific antibody with a brand new sequence, belonging to the technical field of molecular immunology. The CDR-H1 of the heavy chain variable region of the antibody is the amino acid sequence shown in SEQ ID NO. 1, the CDR-H2 is the amino acid sequence shown in SEQ ID NO. 2, and the CDR-H3 is the amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the variable region of the light chain of the antibody is an amino acid sequence shown in SEQ ID NO. 4. The bispecific antibody Vs3P4 can effectively bind to PD-1 and VEGF proteins, effectively compete with PDL-1 for binding to PD-1 protein and compete with VEGF-A for binding to VEGF protein, and simultaneously can effectively stimulate T cell function and secrete cytokines IL-2 and IFN-gamma, while the isotype control antibody can not promote T cell proliferation and IL-2 and IFN-gamma secretion, and in addition, the bispecific antibody Vs3P4 can obviously inhibit mouse tumor growth.

Description

anti-VEGF-anti-PD 1 bispecific antibody with brand new sequence

Technical Field

the invention relates to an anti-VEGF-anti-PD-1 bispecific antibody Vs3P4 with a brand-new sequence, belonging to the technical field of molecular immunology.

Background

Vascular Endothelial Growth Factor (VEGF), also known as Vascular Permeability Factor (VPF), is a highly specific vascular endothelial cell growth factor that has the effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. Vascular endothelial growth factor is a family, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and Placental Growth Factor (PGF). VEGF is commonly known as VEGF-A. VEGF-A promotes neovascularization and increases vascular permeability, VEGF-B plays a role in non-neovascularization tumors, VEGF-C and VEGF-D play a role in the formation of neovascularization and neolymphangiogenesis in cancer tissues, VEGF-E is also a potential neovascularization factor, PGF promotes neovascularization, increases vascular permeability, and PGF expression in experimental choroidal neovascularization is significantly increased. The high affinity receptor that specifically binds to vascular endothelial growth factor is called Vascular Endothelial Growth Factor Receptor (VEGFR), and is mainly classified into 3 types VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-1 and VEGFR-2 are mainly distributed on the surface of tumor vascular endothelium and regulate the generation of tumor blood vessels; VEGFR-3 is distributed mainly on the surface of lymphatic endothelium and regulates the generation of tumor lymphatic vessels. VEGF is a highly conserved homodimeric glycoprotein. Two single chains each having a molecular weight of 24kDa form a dimer with disulfide bonds. VEGF-decomposing monomers are inactive and removal of the N2 glycosyl has no effect on biological effects, but may play a role in cellular secretion. At least 5 protein forms of VEGF121, VEGF145, VEGF165, VEGF185, VEGF206 and the like are produced due to different shearing modes of mRNA, wherein VEGF121, VEGF145 and VEGF165 are secreted soluble proteins and can directly act on vascular endothelial cells to promote the proliferation of the vascular endothelial cells and increase the vascular permeability. In 1990, Philippine Folkman, university of Harvard, USA, proposed a well-known Folkman theory, that is, tumor tissue growth, which must rely on neovascularization to provide sufficient oxygen and nutrients to sustain. Is considered to be the basis for the clinical application of VEGF. The monoclonal antibody of anti-VEGF and VEGFR can inhibit vascular endothelial growth factor, and can be used for treating various metastatic cancers.

Programmed death receptor 1 (PD-1), an important immunosuppressive molecule, is an immunoglobulin superfamily, is a membrane protein of 268 amino acid residues. It was originally cloned from the apoptotic mouse T cell hybridoma 2B 4.11. The immunoregulation taking PD-1 as a target point has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like. The ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. PD-1 and PD-L1 bind to initiate programmed death of T cells, allowing tumor cells to gain immune escape. PD-1 has at least two ligands, one is PD-L1 and one is PD-L2; PD-L1 has at least two ligands, one is PD-1 and one is CD 80; PD-L2 has at least two ligands, one PD-1 and one RGMB. PD-L1/L2 is expressed in antigen presenting cells, and PD-L1 is also expressed in various tissues. Binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, regulating T cell activation and proliferation, and acting like a negative regulator of CTLA-4. The chinese scientist shoji laboratory first found that PD-L1 is highly expressed in tumor tissue and modulates the function of tumor-infiltrating CD8T cells. Therefore, the immunoregulation taking PD-1/PD-L1 as a target has important significance for resisting tumors. In recent years, a plurality of anti-PD-1/PD-L1 antibodies have been rapidly developed in clinical research of tumor immunotherapy. Pembrolizumab and Nivolumab are currently FDA approved for advanced melanoma, and Nivolumab has recently also been FDA approved in the United states for treatment of advanced squamous non-small cell lung cancer. In addition, MPDL3280A (anti-PD-L1 mab), Avelumab (anti-PD-L1 mab), and the like have also entered several advanced clinical studies covering multiple tumor species such as non-small cell cancer, melanoma, bladder cancer, and the like. Due to the broad anti-tumor prospects and surprising potency of PD-1 antibodies, it is widely accepted in the industry that antibodies directed against the PD-1 pathway will bring about breakthrough advances in the treatment of a variety of tumors: can be used for treating non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma, leukemia, anemia, etc. After the clinical efficacy data on PD-1 antibody drugs revealed in the american cancer association (AACR) annual meeting in 2012 and 2013 and in the american clinical oncology Association (ASCO) annual meeting, PD-1 antibody became the most popular in-research antibody drug in the pharmaceutical industry worldwide.

A diabody is a bispecific antibody, which is a non-natural antibody whose two arms that bind to an antigen have different specificities. The construction of bifunctional antibodies usually employs biological methods and chemical cross-linking methods, and with the development of antibody engineering and molecular biology techniques, a new method for constructing bifunctional antibodies, i.e., genetic engineering method, has been developed in recent years. The genetic engineering method can construct multifunctional and multipurpose bifunctional antibody and humanized bifunctional antibody. The bifunctional antibody has potential application value in clinical treatment as a novel secondary targeting system. A bispecific antibody Blincyto (Blinatumomab) developed by FDA approval ann company in usa at 03 th 12 th 2014 is marketed for the treatment of acute lymphocytic leukemia. Blinatumomab is a CD19, CD3 bispecific antibody, Blincyto (Blinatumomab) is the first bispecific antibody approved by FDA in the united states. At present, more than 40 bifunctional antibody formats have been demonstrated to exist, but the development of bispecific antibodies has been difficult due to problems such as low production efficiency and poor pharmacokinetic properties.

The Chinese invention patent application No. 2015106924845.5, the patent name "an anti-VEGF-anti-PD-1 bifunctional antibody and the application thereof" provides an anti-VEGF-anti-PD-1 bifunctional antibody, which is formed by combining a PD1 antibody as a framework and a VEGF antibody as a single chain. The invention optimizes the structure and sequence based on the bifunctional antibody.

Disclosure of Invention

The invention aims to provide a stable and brand-new anti-VEGF-anti-PD 1 bispecific antibody Vs3P4, which has high affinity and high specificity, can specifically recognize two targets of VEGF and PD1, and overcomes the defects that the existing antibody has single effect and cannot adapt to complex diseases and the like.

The invention is realized by the following technical scheme:

An anti-VEGF-anti-PD 1 bispecific antibody Vs3P4 with a brand-new sequence, wherein a CDR-H1 of a heavy chain variable region of the antibody is an amino acid sequence shown in SEQ ID NO. 1, a CDR-H2 is an amino acid sequence shown in SEQ ID NO. 2, and a CDR-H3 is an amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the variable region of the light chain of the antibody is an amino acid sequence shown in SEQ ID NO. 4.

preferably, the CDR-H1 of the heavy chain variable region of the antibody is the nucleotide sequence shown in SEQ ID NO.5, the CDR-H2 is the nucleotide sequence shown in SEQ ID NO. 6, and the CDR-H3 is the nucleotide sequence shown in SEQ ID NO. 7; and the CDR-L of the variable region of the light chain of the antibody is the nucleotide sequence shown in SEQ ID NO. 8.

Preferably, the heavy chain constant region sequence of the antibody is that of human IgG1 and the light chain constant region sequence is that of a human kappa antibody light chain constant region.

Preferably, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO 9.

Preferably, the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 10.

Preferably, the heavy chain nucleotide sequence of the antibody is shown as SEQ ID NO. 11.

Preferably, the light chain nucleotide sequence of the antibody is shown as SEQ ID NO. 12.

A pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

The use of the above antibody in the preparation of a medicament for inhibiting or neutralizing the activity of VEGF and PD 1.

Preferably, the medicament inhibiting or neutralizing VEGF and PD1 activity is for use in the treatment of cancer.

The invention has the beneficial effects that:

The bispecific antibody Vs3P4 can effectively bind to PD-1 and VEGF proteins, effectively compete with PDL-1 for binding to PD-1 protein and compete with VEGF-A for binding to VEGF protein, and simultaneously can effectively stimulate T cell function and secrete cytokines IL-2 and IFN-gamma, while the isotype control antibody can not promote T cell proliferation and IL-2 and IFN-gamma secretion, and in addition, the bispecific antibody Vs3P4 can obviously inhibit mouse tumor growth.

Drawings

FIG. 1 is a diagram showing the results of SDS-PAGE electrophoresis of PD-1 and VEGF antigens (wherein A is VEGF antigen; B is PD-1 antigen);

FIG. 2 is a diagram showing the results of electrophoretic detection of an anti-PD-1 humanized antibody PDAB;

FIG. 3 is a diagram showing the result of electrophoretic detection of the anti-VEGF humanized antibody Avastin;

FIG. 4 is a diagram showing the results of the electrophoretic detection of bispecific antibody A3P 4;

Figure 5 is a graph of SEC detection results for bispecific antibody A3P 4;

Figure 6 is a schematic protein structure diagram of bispecific antibody Vs3P 4;

FIG. 7 is a graph showing the results of electrophoretic detection of bispecific antibody Vs3P 4;

Figure 8 is a graph of SEC detection results for bispecific antibody Vs3P 4;

FIG. 9 is a schematic diagram of the protein structure of the bispecific antibody Ps3 Vm;

FIG. 10 is a graph showing the results of electrophoretic detection of bispecific antibody Ps3 Vm;

Fig. 11 is a graph of SEC detection results for bispecific antibody Ps3 Vm;

FIG. 12 is a graph comparing the relative binding activity of PDAB, A3P4, Vs3P4, Ps3Vm to PD1-His in ELISA;

FIG. 13 is a graph comparing the relative binding activity of Avastin, A3P4, Vs3P4, Ps3Vm to rHuVEGF by ELISA;

FIG. 14 is a competition ELISA to identify the specificity of the binding epitope of PDAB, A3P4, Vs3P4, Ps3Vm, Avastin and PD 1;

FIG. 15 is a competition ELISA to identify the specificity of the binding epitopes of PDAB, A3P4, Vs3P4, Ps3Vm, Avastin and VEGF;

FIG. 16 is a graph of the amount of IL-2 secretion induced by Nivolumab, PDAB, Vs3P4, Ps3Vm, and IgG1 in vitro by T cells as a function of antibody concentration;

FIG. 17 is a graph of the amount of IFN-. gamma.secretion induced by Nivolumab, PDAB, Vs3P4, Ps3Vm, and IgG1 in vitro by T cells as a function of antibody concentration;

FIG. 18 is a graph showing the weight change of a preliminarily constructed mouse model;

FIG. 19 is a graph showing the change in tumor volume of a mouse model constructed preliminarily.

Detailed Description

For a better understanding of the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting thereof. The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available.

Example 1 preparation of PD1 and VEGF antigen, antibody

1. Expression vector construction of PD-1 antigen

The cDNA of PD-1 of human is synthesized from Nanjing Kingsler company, the GeneID is 5133, the cDNAID is NM-005018.2, the PD-1-mFc is obtained by adding Fc purification label after synthesizing the PD-1 gene of extracellular region, Xba I is introduced at both ends, two restriction enzyme cutting sites of Bam H I are connected to pTT5 expression plasmid, and the correctness is verified by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanjin Biotechnology Ltd.), and a single clone was selected and inoculated into 1L of LB liquid medium to OD₆₀₀At 1, the cells were collected by centrifugation and plasmids were extracted using a plasmid macroextraction kit (purchased from Qiagen).

2. expression vector construction of VEGF antigen

The amino acid corresponding to the Gene VEGF (NCBI Gene ID:7422) and the Fc protein fragment mFc (Ig gamma-2A chain C region) of mouse IgG are fused and designed to obtain VEGF-mFc. In order to improve the expression efficiency of the target gene in a 293F cell expression system, the sequence is optimized, two restriction enzyme sites of Xba I and Bam H I are introduced at two ends and are connected to a pTT5 expression plasmid, and the correctness is verified by sequencing. Sequenced plasmid transfection Trans10 (from Beijing Kogyo gold Biotech Co., Ltd.), selecting a single clone, inoculating to 1 liter of LB liquid medium to OD₆₀₀At 1, the cells were collected by centrifugation and plasmids were extracted using a plasmid macroextraction kit (purchased from Qiagen).

3. Expression and purification of PD-1 and VEGF antigens

Expression vectors identified by sequencing as correct were transfected into 293F cells (purchased from Invitrogen), 37 degrees, 5% CO₂After culturing at 130rpm/min for 7 days, the supernatant was collected by centrifugation. Centrifuging the supernatant at 4000rpm for 10min, and filtering with 0.45 μm filter membrane; adding 400mM NaCl into the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, it was loaded into PBS (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄pH7.4) 5mL balanced HiTrap Protein A column; after the sample is completely loaded, the sample is washed by PBS, the flow rate is 5mL/min, and the ultraviolet monitoring is horizontal. Buffer B (1M Glycine, pH3.5) was eluted at a flow rate of 1mL/min, and the collected effluent peaks were neutralized to pH7.5 with Tris-PAGE and examined by SDS-PAGE, the results of which are shown in FIG. 1. The elution peak was concentrated into PBS using an ultrafiltration concentration tube, thereby obtaining an antigen.

4. Construction of anti-PD 1 humanized antibody

(1) Antigen-immunized mice and hybridoma screening

3 female BALB/c mice of 8 weeks old are selected for the experiment, and the mice are immunized by mixing PD-1 extracellular region antigen and Freund's complete adjuvant 1 time a week and 3 times in total by adopting an intraperitoneal injection method. Measuring the serum titer of the mice one week after the last immunization, strengthening the immunization once after the condition titer is more than 8K is met, and the result shows that 3 mice completely meet the titer (the titer of the antibody is determined by the dilution value corresponding to the OD450 value which is more than 2 times of the negative control and more than 0.25, and the titer is more than or equal to 8K, so that the requirement is met), killing the mice after 3 days, taking the spleen of the mice, and grinding to obtain the splenocytes. The results of the ELISA assay for serum titers in mice are shown in Table 1.

TABLE 120871 mouse immune serum ELISA assays

B cells of the anti-human PD-1 antibody were selected by flow cytometry (FACS), and the selected B cells were placed in RPMI1640 medium, myeloma cells (SP2/0) were added thereto and mixed well, and cell fusion was carried out with 50% PEG solution. The fused cells are diluted properly, cultured in multiple 96-well culture plates, HAT selective medium is added to kill unfused B cells and myeloma cells, and hybridoma cells are obtained. Collecting the cell culture supernatant of the 96-well plate after culturing for 2 weeks, combining the cell culture supernatant with a 96-well enzyme label plate paved with PD-1 antigen for 1 hour, adding an anti-mouse/HRP secondary antibody for incubation for 1 hour, finally adding a TMB color reagent for reaction for 10 minutes, measuring the light absorption value at 450nm by using an enzyme label instrument, and selecting the hybridoma cells with the binding activity with PD-1 (primary screening: 12 96-well plates, and obtaining 42 holes with OD value more than or equal to 0.5). Followed by flow cytometry (FACS) screening to select hybridoma cells with PD-1/PD-L1 blocking activity. And then carrying out subcloning by a limiting dilution method, culturing the cells subjected to limiting dilution into a 96-well plate, marking the monoclonal and the polyclonal when the clones grow to 1/6 of the whole well, and carrying out ELISA detection on the monoclonal. After detection, the monoclonal antibody with the highest OD value is subjected to limiting dilution and then is inoculated into a 96-well plate, and is subjected to subcloning again as described above, the process is repeated for a plurality of times until the positive well ratio is 100%, the strain is successfully established, and finally the anti-PD-1 murine monoclonal antibody cell strain is obtained, wherein the subcloning result of the limiting dilution method is shown in table 2, and the affinity identification result is shown in table 3.

TABLE 2 Positive cloning well plate sites

Serial number	Positive clones	96-well plate position	384 orifice plate position	OD value
					1	2G8-1N8	2G8	1N8	1.022

TABLE 3 affinity identification

(2) anti-PD-1 murine antibody variable region Gene calling

Selecting anti-PD-1 hybridoma clone, extracting total RNA by adopting a Trizol method, carrying out reverse transcription PCR by utilizing an antibody subtype (Isotype) specific primer or a universal primer, respectively amplifying genes of a light chain variable region (VL) and a heavy chain variable region (VH) of an antibody, and then connecting to a cloning vector for DNA sequencing analysis. Finally, the complete DNA sequences of VL and VH are obtained and translated into the corresponding amino acid sequences. The amino acid sequences of the heavy chain and the light chain of the anti-PD-1 murine antibody are respectively SEQ ID NO 13-14; wherein, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are respectively SEQ ID NO. 15-17, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are respectively SEQ ID NO. 18-20.

(3) anti-PD-1 murine monoclonal antibody variable region gene humanization transformation

(a) humanization of heavy chains

Human germline genes (germline genes) with higher homology to the VH gene of the mouse PD-1 antibody were first analyzed using Ig Blast (http:// www.ncbi.nlm.nih.gov/igblast). The results showed that heavy chain IGHV3-23 has 83% homology at the amino acid level and was therefore selected as a candidate gene template for the heavy chain variable region. The CDR-H1, CDR-H2, and CDR-H3 of the mouse PD-1 antibody were numbered according to Kabat numbering convention, and the corresponding CDR region amino acid sequences were introduced into the framework regions of IGHV 3-23. The amino acids No.49(S- > T) and No.78(T- > N) of the framework region are back mutated into the original sequence of the mouse PD-1 antibody. Then, heavy chain CDRs H1No.33(G- > D), H2No.56(S- > R) were additionally mutated, thereby completing the humanization of the heavy chain variable region. The heavy chain amino acid sequence of the anti-PD-1 humanized antibody is SEQ ID NO. 21; wherein the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are respectively SEQ ID NO. 22-24.

(b) Humanization of light chains

Human germline genes with higher homology to the VL gene of mouse PD-1 antibodies were first analyzed using Ig Blast (http:// www.ncbi.nlm.nih.gov/igblast). The results showed that light chain IGKV1-16 has 86% homology at the amino acid level and was therefore selected as a candidate gene template for the light chain variable region. The CDR-L1, CDR-L2 and CDR-L3 of the mouse PD-1 antibody were numbered according to the Kabat numbering convention, and the corresponding CDR region amino acid sequences were introduced into the framework regions of IGKV 1-16. The framework region amino acid No.83(F- > M) was back mutated to the original sequence of the mouse PD-1 antibody. Then, light chain CDRs L1No.31(S- > T) and 34(S- > A), L2No.56(D- > L) were additionally mutated, thereby completing the light chain variable region humanization. The light chain amino acid sequence of the anti-PD-1 humanized antibody is SEQ ID NO. 25; wherein the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are respectively SEQ ID NO: 26-28.

(4) Affinity maturation of anti-PD-1 humanized antibodies

antibody mutant libraries were designed for 5 CDR regions (L1, L3, H1, H2 and H3) of the anti-PD-1 humanized antibody, respectively, with the mutation sites covering non-conserved sites of all CDR regions. Obtaining single chain antibody (scFv) genes by adopting SOE-PCR reaction, recovering DNA gel, carrying out enzyme digestion, connecting with a pCANTAB-5E phage display carrier after enzyme digestion, and electrically transforming TG1 competent bacteria to obtain 5 single chain antibody libraries containing CDR mutation. Recombinant phages were prepared by infecting M13KO7 helper phage, and three rounds of panning were performed in total to retain and enrich for antibody mutants with strong binding capacity. And (3) respectively combining the recombinant phage with biotin-labeled recombinant human PD-1 antigen for 2 hours in each round of panning, then adding streptavidin magnetic beads for combination for 30 minutes, washing the phage for 5 times with 2% TPBS, 1% TPBS and PBS (phosphate buffer solution) in sequence, and infecting TG1 cells immediately after panning for preparing the recombinant phage in the next round, wherein each time is 5 minutes. Selecting three rounds of elutriated enriched TG1 monoclonals, preparing recombinant phage supernatant, combining with a 96-hole enzyme label plate paved with 1 mu g/mL PD-1 antigen for 1 hour, adding M13/HRP secondary antibody for incubation for 1 hour, finally adding OPD for color reaction for 10 minutes, and measuring the light absorption value at 490nm by using an enzyme label instrument. After data are analyzed, the relative affinities of the antibody-containing mutants are calculated, 3, 6 and 5 clones with obviously improved affinities are respectively screened from L3, H1 and H3 mutant libraries, and finally 1 clone PDAB with the highest affinity is selected from the H3 mutant library to carry out the next research, wherein the electrophoresis result is shown in FIG. 2.

5. Construction of anti-VEGF humanized antibodies

The anti-VEGF humanized antibody used in the experiment is bevacizumab (Avastin, bevacizumab) marketed by Roche (Genentech) in 2004, an antibody sequence (CN101210051A) is obtained from a protein sequence website disclosed by patent website and the like, cDNAs of a light chain and a heavy chain of a VEGF antibody are artificially synthesized, the synthesized cDNAs are respectively cloned into pTT5 plasmid, and the correct construction of the plasmid is determined by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanjin Biotechnology Ltd.), and a single clone was selected and inoculated into 1L of LB liquid medium to OD₆₀₀At 1, the cells were collected by centrifugation and plasmids were extracted using a plasmid macroextraction kit (purchased from Qiagen). Sequencing-identified correct VEGF heavy and light chain expression vectors (1:1) were CO-transfected into 293F cells at 37 ℃ and 5% CO₂After culturing at 130rpm/min for 7 days, the supernatant was collected by centrifugation. Centrifuging the supernatant at 4000rpm for 10min, filtering with 0.45 μm filter membrane, and collecting filtrate; adding 400mM NaCl into the filtrate; the pH was adjusted to 8.0. After the sample was filtered again through a 0.2 μm filter, it was loaded into PBS (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mMKH₂PO₄Ph7.4) 5mL HiTrap MabSelect column (from GE); after the sample is completely loaded, the sample is washed by PBS, the flow rate is 5mL/min, and the ultraviolet monitoring is horizontal. Buffer B (1M Glycine, pH3.5) elution, flow rate of 1mL/min, collection of the outflow peak with Tris neutralized to pH7.5, and SDS-PAGE detection, SDS-PAGE non-reduction electrophoresis detection results are shown in figure 3. Concentrating the eluate with ultrafiltration concentrating tube, and exchanging the eluate with desalting columnInto PBS, thereby obtaining the antibody VEGF protein.

Example 2 preparation of candidate bispecific antibodies

Preparation of scFv-VEGF-linker-PD1-H chain structure bispecific antibody (A3P 4):

On the basis of the existing anti-VEGF humanized antibody, the heavy chain and light chain variable region genes are extracted and connected into a single-chain antibody scFv-VEGF by peptide segments. Cloning scFv-VEGF into the N end of the heavy chain of the anti-PD 1 antibody to construct the bispecific antibody with scFv-VEGF-linker-PD1-Hchain structure. The heavy chain expression vector and the light chain expression vector of the anti-PD 1 antibody are co-transformed into 293F cells, supernatant is collected and purified, SDS-PAGE identifies molecular weight and purity (figure 4), and SEC detects that the sequence has more antibody dimers (figure 5).

Preparation of dsFv-VEGF-linker-PD1-H chain structure bispecific antibody (Vs3P 4):

On the basis of the original experiment, a new structure is redesigned. The heavy chain and light chain variable region genes of the anti-VEGF humanized antibody are extracted, VH44cys and VL100cys mutations (increasing intra-chain disulfide bonds to improve aggregation) are carried out, and peptide chains are used for connecting into a single-chain antibody dsFv-VEGF. The dsFv-VEGF was cloned to the N-terminus of the anti-PD 1 antibody heavy chain to construct a bispecific antibody of dsFv-VEGF-linker-PD1-Hchain structure (FIG. 6). The heavy chain expression vector and the light chain expression vector of the anti-PD 1 antibody were co-transformed into 293F cells, and the supernatant was collected, purified, and subjected to SDS-PAGE to identify molecular weight and purity (FIG. 7), and SEC detection (FIG. 8). The heavy chain amino acid and nucleotide sequences of the Vs3P4 antibody are respectively SEQ ID NO 9 and SEQ ID NO 11, and the amino acid and nucleotide sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are respectively SEQ ID NO 1-3 and SEQ ID NO 5-7; the light chain amino acid and nucleotide sequences of the Vs3P4 antibody are SEQ ID NO 10 and SEQ ID NO 12, respectively, and the amino acid and nucleotide sequences of the CDR-L of the light chain variable region are SEQ ID NO 4 and SEQ ID NO 8, respectively.

preparation of dsFv-PD1-linker-VEGF-H chain structure bispecific antibody (Ps3 Vm):

And a third structural optimization design, on the basis of the existing anti-PD 1 humanized antibody, extracting heavy chain and light chain variable region genes, carrying out VH44cys and VL100cys mutation (increasing intra-chain disulfide bonds to improve aggregation), and connecting a peptide chain into a single-chain antibody dsFv-PD 1. The dsFv-PD1 was cloned into the N-terminus of the anti-VEGF antibody heavy chain to construct a bispecific antibody with the structure of dsFv-PD1-linker-VEGF-H chain (FIG. 9). The heavy chain expression vector and the light chain expression vector of the anti-VEGF antibody were co-transformed into 293F cells, the supernatant was collected, purified, and characterized for molecular weight and purity by SDS-PAGE (FIG. 10), and SEC detection was performed (FIG. 11).

Example 3 bispecific antibody affinity assay

1. Affinity of bispecific antibodies to PD-1

The ELISA plate is coated with PD-1-mFc, 1% BSA is used for blocking, antibodies PDAB, A3P4, Vs3P4 and Ps3Vm with different concentrations are respectively added into the ELISA plate, after incubation at 37 ℃, enzyme-labeled secondary antibody is added for incubation at 37 ℃ for 30 minutes. And detecting the light absorption value of 450nm on a microplate reader. The results of the binding of the antibodies PDAB, A3P4, Vs3P4, Ps3Vm to the antigen PD-1 show that the antibodies PDAB, A3P4, Vs3P4, Ps3Vm all bind efficiently to the PD-1 protein, and that the binding efficiency is dose-dependent, and the results are shown in fig. 12 and table 4.

TABLE 4 binding efficiency of antibodies PDAB, A3P4, Vs3P4, Ps3Vm to PD-1 protein

2. Affinity of bispecific antibodies to VEGF

VEGF-mFc is used for coating an enzyme label plate, 1% BSA is used for blocking, antibodies Avastin, A3P4, Vs3P4 and Ps3Vm with different concentrations are respectively added into the enzyme label plate, after incubation at 37 ℃, enzyme-labeled secondary antibody is added for incubation at 37 ℃ for 30 minutes. And detecting the light absorption value of 450nm on a microplate reader. The results of the binding of the antibodies Avastin, A3P4, Vs3P4, Ps3Vm to the antigen VEGF show that the antibodies Avastin, A3P4, Vs3P4, Ps3Vm all bind to VEGF protein efficiently and that the binding efficiency is dose-dependent, and the results are shown in fig. 13 and table 5.

TABLE 5 binding efficiency of the antibodies Avastin, A3P4, Vs3P4, Ps3Vm to the VEGF protein

Example 4 bispecific antibody specificity assay

1. Specificity of bispecific antibodies against PD-1

an enzyme label plate is coated by PD-1-mFc, 1% BSA is used for blocking, antibodies PDAB, A3P4, Vs3P4, Ps3Vm and Avastin with different concentrations are mixed with PDL-1-hFc respectively, and after incubation at 37 ℃, enzyme-labeled secondary antibody is added for incubation at 37 ℃ for 30 minutes. And detecting the light absorption value of 450nm on a microplate reader. The results of the binding of the antibodies PDAB, A3P4, Vs3P4, Ps3Vm and Avastin to the antigen PD-1 show that the antibodies PDAB, A3P4, Vs3P4, Ps3Vm and Avastin can effectively compete with PDL-1 to bind to the PD-1 protein, and the binding efficiency is dose-dependent, and the results are shown in FIG. 14.

2. Specificity of bispecific antibodies against VEGF

VEGF-mFc is used for coating an enzyme label plate, 1% BSA is used for blocking, antibodies Avastin, A3P4, Vs3P4, Ps3Vm and PDAB with different concentrations are mixed with VEGF-A-hFc respectively, and after incubation at 37 ℃, enzyme-labeled secondary antibody is added for incubation at 37 ℃ for 30 minutes. And detecting the light absorption value of 450nm on a microplate reader. The results of the binding of the antibodies Avastin, A3P4, Vs3P4, Ps3Vm and PDAB to the antigen VEGF show that the antibodies Avastin, A3P4, Vs3P4, Ps3Vm and PDAB can compete effectively with VEGF-a for binding to VEGF protein, and the binding efficiency is dose-dependent, and the results are shown in fig. 15.

Example 5 in vitro Induction of IL-2 secretion by candidate bispecific antibodies

Human T cells were purified by preparing fresh PBMC using Ficoll centrifugation (from GE) and CD4+ T cell enrichment columns (from R & D Systems). Cells were plated in 96-well flat-bottom plates, after overnight incubation, six different concentrations of antibodies NIVO, PDAB, Vs3P4, Ps3Vm were added at 0.0096, 0.048, 0.24, 1.2, 6, 30. mu.g/mL, and isotype control antibody IgG1 at the same six concentrations was added as a negative control, and after 3 days of incubation, the supernatant was collected and assayed for IL-2 secretion by a Luminex apparatus (from Life technology) and a cytokine IL-2 detection kit (from BD Biosciences). The results are shown in fig. 16, and show that: bispecific antibodies Vs3P4 and Ps3Vm both effectively stimulate T cell function and secrete the cytokine IL-2, and are related to antibody concentration, whereas isotype control antibodies do not promote T cell proliferation and IL-2 secretion.

example 6 candidate bispecific antibody induces T cells to secrete IFN- γ in vitro

Fresh PBMC were prepared and human T cells were purified by Ficoll centrifugation (from GE) and CD4+ T cell enrichment column (from R & D Systems). Monocytes were purified using the Miltenyi CD14 monocyte purification kit and DC cells were generated after 7 days of monocyte culture with GM-CSF and IL-4 (both purchased from PeproTech). Cells were plated into 96-well flat-bottom plates and after overnight culture, each culture contained 10e5 purified T cells and 10e4 dendritic cells in a total volume of 200 μ Ι _. Six different concentrations of antibodies NIVO, PDAB, Vs3P4, Ps3Vm were added at 0.0096, 0.048, 0.24, 1.2, 6, 30. mu.g/mL, and six concentrations of isotype control antibody IgG1 were added as negative controls. The cells were cultured at 37 ℃ for 5 days. After 5 days, 100. mu.L of medium was removed from each culture for cytokine IFN-. gamma.measurement. The level of IFN-. gamma.was determined using the OptEIA ELISA kit (from BD Biosciences). The results are shown in FIG. 17: bispecific antibodies Vs3P4 and Ps3Vm both effectively stimulate the function of T cells to secrete the cytokine IFN- γ, and are concentration dependent, whereas isotype control antibodies do not promote T cell proliferation and IFN- γ secretion.

Example 7 candidate bispecific antibody inhibits mouse tumor growth

1. initially constructing a mouse model, and selecting PBMC cells suitable for experiments

PBMC cells, human colon cancer Colo-205 cells and B-NDG mice adopted in the experiment are all common types in the market.

Human colon cancer Colo-205 cells purchased from Chinese academy of sciences were cultured to 6.0 x 10⁷Above, B-NDG mice (2.0X 10 each) purchased from Baioselta corporation were inoculated subcutaneously⁶One cell, 30 mice total). Mice were normally bred and tumors were grown to 100mm³At size, human PBMC cells from different sources were purchased at 1 x 10⁷Each abdominal cavity was injected into B-NDG heavily immunodeficient mice purchased from Baioecker, and tumor growth was observed until successful tumorigenesis was achieved (10 PBMC cells selected and 3 mice injected per group)parallel experiments were performed).

The results of the experiment are shown in table 6 below and fig. 18 and 19 (average numbers in the graph):

TABLE 6 preliminary constructed mouse model body weight and tumor volume changes

2. Construction of animal models with selected PBMC cells

PBMC cells (G1, G2, G8 and G10) successfully matched as described above were selected and injected into mice with severe immunodeficiency (1 × 10 each) obtained by knocking out MHC from B-NDG-B2m (Pogostember)⁷Individual cells) and simultaneously subcutaneously inoculated with human colon cancer Colo-205 cells to observe whether the cells had successfully formed tumors. This was a preliminary experiment, and 8 mice were inoculated (2 parallel experiments were performed) and observed to be tumorigenic. And selecting PBMC cells which successfully form tumors to carry out the next stage of experiment.

Tumor volume changes are shown in table 7 below (mean numbers in table): tumor Voiume (mm)³)

Table 7 animal model tumor volume changes constructed from selected PBMC cells

DAY	0	3	7	10	15	18	21
								G1	100.23	230.56	548.73	668.39	1268.45	1684.54	2025.68
G2	108.35	203.21	502.72	851.94	1054.26	1563.81	1954.29
								G8	113.51	211.55	465.84	712.43	946.25	1458.48	1743.65
G10	100.62	198.36	600.26	900.25	1356.31	1965.32	2200.25

3. Construction of animal model for experiment

The PBMC cells (G10) were selected and injected into MHC-knocked-out B-NDG-B2m severe immunodeficiency mice (1 × 10 each) from Baioensi⁷4 cells in total, 6 mice in each group), and simultaneously inoculating human colon cancer Colo-205 cells subcutaneously to observe whether the cells are successfully tumorigenic. Mice were randomized into 4 groups according to tumor growth. A negative control group (intraperitoneal injection of physiological saline), a Vs3P4 group (tail vein injection of Vs3P4 antibody 3mg/kg), a Ps3Vm group (tail vein injection of Ps3Vm antibody 3mg/kg), and a positive control group of bevacizumab (tail vein injection of 3 mg/kg). The administration was 1 time every 3 days for a total of 21 days. Tumor volume changes are as follows: tumor Voiume (mm)³)

TABLE 8 Experimental animal model tumor volume changes

Number of days	0	3	7	10	15	18	21
								Physiological saline	400.23	609.56	812.26	1009.32	1478.26	1993.54	2365.82
Vs3P4	403.26	454.35	353.02	256.51	202.56	194.32	203.45
								Ps3Vm	400.24	469.35	260.24	134.87	108.46	156.36	147.51
Avastin antibodies	408.58	498.69	338.56	305.45	289.5	306.43	324.62

The invention tests 3 anti-VEGF-anti-PD 1 bispecific antibodies with different structures, respectively verifies the antibody effect from molecule, cell and animal level, and the result shows that: the bispecific antibody Vs3P4 can effectively bind to PD-1 and VEGF proteins, effectively compete with PDL-1 for binding to PD-1 protein and compete with VEGF-A for binding to VEGF protein, and simultaneously can effectively stimulate T cell function and secrete cytokines IL-2 and IFN-gamma, while the isotype control antibody can not promote T cell proliferation and IL-2 and IFN-gamma secretion, and in addition, the bispecific antibody Vs3P4 can obviously inhibit mouse tumor growth.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Han Hai Boxing Biotechnology Limited in Anhui

<120> an anti-VEGF-anti-PD 1 bispecific antibody with a novel sequence

<130> 2019

<160> 28

<170> PatentIn version 3.3

<210> 1

<211> 107

<212> PRT

<213> Artificial sequence

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105

<210> 2

<211> 123

<212> PRT

<213> Artificial sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 118

<212> PRT

<213> Artificial sequence

<400> 3

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Arg Tyr Gly Glu Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 4

<211> 107

<212> PRT

<213> Artificial sequence

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 5

<211> 321

<212> DNA

<213> Artificial sequence

<400> 5

gacatccaga tgacccagag cccaagctcc ctgtctgcca gcgtgggcga ccgggtgacc 60

atcacatgct ccgcctctca ggatatctcc aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaaggtgct gatctacttc acctctagcc tgcacagcgg agtgccctcc 180

agattcagcg gctccggctc tggcacagac tttaccctga caatctcctc tctgcagcca 240

gaggatttcg ccacctacta ttgccagcag tatagcacag tgccctggac ctttggctgc 300

ggcaccaagg tggagatcaa g 321

<210> 6

<211> 369

<212> DNA

<213> Artificial sequence

<400> 6

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc tctgaggctg 60

agctgtgcag catccggata caccttcaca aactatggca tgaattgggt gagacaggca 120

cctggcaagt gcctggagtg ggtgggctgg atcaacacct acacaggcga gccaacatat 180

gccgccgact ttaagcggag attcaccttt tctctggata caagcaagtc caccgcctac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt actattgcgc caagtaccct 300

cactactatg gcagctccca ctggtatttc gacgtgtggg gccagggcac actggtgacc 360

gtgtctagc 369

<210> 7

<211> 354

<212> DNA

<213> Artificial sequence

<400> 7

caggtgcagc tggtggagag tggaggagga ctggtccagc ctggaggctc tctgagactg 60

tcctgcgcag catccggatt cgccttttcc tcttacgaca tgtcctgggt gaggcaggca 120

ccaggcaagg gcctggagtg ggtagcaaca atctctggag gcggccggta cacctactat 180

cccgacagcg tgaagggcag gtttaccatc tctcgcgata acagcaagaa caatctgtat 240

ctgcagatga atagcctgcg ggccgaggat acagccgtgt actactgtgc cgtgagatac 300

ggcgagacct ggttcgccta ttggggccag ggtaccctgg tgaccgtgag ctcc 354

<210> 8

<211> 321

<212> DNA

<213> Artificial sequence

<400> 8

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60

atcacctgca gggccagcca ggacatcaac acctacctgg cctggttcca gcagaagccc 120

ggcaaggccc ccaagagcct gatctacagg gccaacaggc tggtgagcgg cgtgcccagc 180

aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacatgg ccacctacta ctgcctgcag tacgacgagt tccccctgac cttcggcgcc 300

ggcaccaagc tggagctgaa g 321

<210> 9

<211> 730

<212> PRT

<213> Artificial sequence

<400> 9

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser

100 105 110

Thr Val Pro Trp Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

145 150 155 160

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu

180 185 190

Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala

195 200 205

Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser

210 215 220

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr

245 250 255

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

275 280 285

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

290 295 300

Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser

305 310 315 320

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile

325 330 335

Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg

340 345 350

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr Leu Gln Met

355 360 365

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg

370 375 380

Tyr Gly Glu Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

385 390 395 400

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

405 410 415

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

420 425 430

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

435 440 445

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

450 455 460

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

465 470 475 480

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

485 490 495

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

500 505 510

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

515 520 525

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

530 535 540

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

545 550 555 560

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

565 570 575

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

580 585 590

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

595 600 605

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

610 615 620

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

625 630 635 640

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

645 650 655

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

660 665 670

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

675 680 685

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

690 695 700

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

705 710 715 720

Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

725 730

<210> 10

<211> 234

<212> PRT

<213> Artificial sequence

<400> 10

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp

35 40 45

Ile Asn Thr Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Ser Leu Ile Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Glu Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 11

<211> 2196

<212> DNA

<213> Artificial sequence

<400> 11

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg atccacaggc 60

gacatccaga tgacccagag cccaagctcc ctgtctgcca gcgtgggcga ccgggtgacc 120

atcacatgct ccgcctctca ggatatctcc aactacctga attggtatca gcagaagccc 180

ggcaaggccc ctaaggtgct gatctacttc acctctagcc tgcacagcgg agtgccctcc 240

agattcagcg gctccggctc tggcacagac tttaccctga caatctcctc tctgcagcca 300

gaggatttcg ccacctacta ttgccagcag tatagcacag tgccctggac ctttggctgc 360

ggcaccaagg tggagatcaa gggaggagga ggctccggcg gaggaggctc tggcggcggc 420

ggcagcggag gcggcggctc cgaggtgcag ctggtggagt ccggcggcgg cctggtgcag 480

cccggcggct ctctgaggct gagctgtgca gcatccggat acaccttcac aaactatggc 540

atgaattggg tgagacaggc acctggcaag tgcctggagt gggtgggctg gatcaacacc 600

tacacaggcg agccaacata tgccgccgac tttaagcgga gattcacctt ttctctggat 660

acaagcaagt ccaccgccta cctgcagatg aacagcctga gggccgagga caccgccgtg 720

tactattgcg ccaagtaccc tcactactat ggcagctccc actggtattt cgacgtgtgg 780

ggccagggca cactggtgac cgtgtctagc ggcggcggcg gctctggagg aggaggcagc 840

ggaggaggag gctcccaggt gcagctggtg gagagtggag gaggactggt ccagcctgga 900

ggctctctga gactgtcctg cgcagcatcc ggattcgcct tttcctctta cgacatgtcc 960

tgggtgaggc aggcaccagg caagggcctg gagtgggtag caacaatctc tggaggcggc 1020

cggtacacct actatcccga cagcgtgaag ggcaggttta ccatctctcg cgataacagc 1080

aagaacaatc tgtatctgca gatgaatagc ctgcgggccg aggatacagc cgtgtactac 1140

tgtgccgtga gatacggcga gacctggttc gcctattggg gccagggtac cctggtgacc 1200

gtgagctccg ctagcacaaa gggaccaagc gtgtttccac tggcaccatg ctctcggagc 1260

acatccgagt ctaccgccgc cctgggatgt ctggtgaagg actacttccc tgagccagtg 1320

accgtgtctt ggaacagcgg cgccctgaca agcggagtgc acacctttcc tgccgtgctg 1380

cagtctagcg gcctgtactc cctgtcctct gtggtgacag tgcccagctc ctctctgggc 1440

accaagacat atacctgcaa cgtggaccac aagccttcca ataccaaggt ggataagagg 1500

gtggagtcta agtacggacc accttgccca ccatgtccag cacctgagtt cctgggagga 1560

ccaagcgtgt tcctgtttcc tccaaagccc aaggacaccc tgatgatctc ccgcacacca 1620

gaggtgacct gcgtggtggt ggacgtgtct caggaggacc ccgaggtgca gttcaactgg 1680

tacgtggatg gcgtggaggt gcacaatgcc aagaccaagc ctagggagga gcagtttaat 1740

tccacatacc gcgtggtgtc tgtgctgacc gtgctgcacc aggattggct gaacggcaag 1800

gagtataagt gcaaggtgag caataagggc ctgccaagct ccatcgagaa gacaatctcc 1860

aaggcaaagg gacagcctcg ggagccacag gtgtacaccc tgccccctag ccaggaggag 1920

atgacaaaga accaggtgtc cctgacctgt ctggtgaagg gcttctatcc ttctgacatc 1980

gccgtggagt gggagagcaa tggccagcca gagaacaatt acaagaccac accacccgtg 2040

ctggactccg atggctcttt ctttctgtat agccggctga cagtggataa gtccagatgg 2100

caggagggca acgtgtttag ctgttccgtg atgcacgagg ccctgcacaa tcactacacc 2160

cagaagtctc tgagcctgtc cctgggcaag taatga 2196

<210> 12

<211> 708

<212> DNA

<213> Artificial sequence

<400> 12

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg atccacaggc 60

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 120

atcacctgca gggccagcca ggacatcaac acctacctgg cctggttcca gcagaagccc 180

ggcaaggccc ccaagagcct gatctacagg gccaacaggc tggtgagcgg cgtgcccagc 240

aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 300

gaggacatgg ccacctacta ctgcctgcag tacgacgagt tccccctgac cttcggcgcc 360

ggcaccaagc tggagctgaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gctaatga 708

<210> 13

<211> 118

<212> PRT

<213> Artificial sequence

<400> 13

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ser Arg Phe Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 14

<211> 107

<212> PRT

<213> Artificial sequence

<400> 14

Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 15

<211> 13

<212> PRT

<213> Artificial sequence

<400> 15

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser

1 5 10

<210> 16

<211> 17

<212> PRT

<213> Artificial sequence

<400> 16

Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 17

<211> 11

<212> PRT

<213> Artificial sequence

<400> 17

Ala Ser Arg Phe Gly Glu Ala Trp Phe Ala Tyr

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence

<400> 18

Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser

1 5 10

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence

<400> 19

Tyr Arg Ala Asn Arg Leu Val Asp

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<400> 20

Leu Gln Tyr Asp Glu Phe Pro Leu Thr

1 5

<210> 21

<211> 118

<212> PRT

<213> Artificial sequence

<400> 21

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 22

<211> 13

<212> PRT

<213> Artificial sequence

<400> 22

Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser

1 5 10

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<400> 23

Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 24

<211> 11

<212> PRT

<213> Artificial sequence

<400> 24

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr

1 5 10

<210> 25

<211> 108

<212> PRT

<213> Artificial sequence

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<400> 26

Arg Ala Ser Gln Asp Ile Asn Thr Tyr Leu Ala

1 5 10

<210> 27

<211> 8

<212> PRT

<213> Artificial sequence

<400> 27

Tyr Arg Ala Asn Arg Leu Val Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<400> 28

Leu Gln Tyr Asp Glu Phe Pro Leu Thr

1 5

Claims

1. An anti-VEGF-anti-PD 1 bispecific antibody of novel sequence, characterized in that: the CDR-H1 of the heavy chain variable region of the antibody is an amino acid sequence shown by SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown by SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown by SEQ ID NO. 3; and the CDR-L of the variable region of the light chain of the antibody is an amino acid sequence shown in SEQ ID NO. 4.

2. The anti-VEGF-anti-PD 1 bispecific antibody of claim 1, which has a novel sequence: the CDR-H1 of the heavy chain variable region of the antibody is the nucleotide sequence shown in SEQ ID NO.5, the CDR-H2 is the nucleotide sequence shown in SEQ ID NO. 6, and the CDR-H3 is the nucleotide sequence shown in SEQ ID NO. 7; and the CDR-L of the variable region of the light chain of the antibody is the nucleotide sequence shown in SEQ ID NO. 8.

3. The anti-VEGF-anti-PD 1 bispecific antibody of claim 1, which has a novel sequence: the heavy chain constant region sequence of the antibody is that of human IgG1, and the light chain constant region sequence is that of human kappa antibody.

4. The anti-VEGF-anti-PD 1 bispecific antibody of claim 1, which has a novel sequence: the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 9.

5. the anti-VEGF-anti-PD 1 bispecific antibody of claim 1, which has a novel sequence: the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 10.

6. A full-new sequence anti-VEGF-anti-PD 1 bispecific antibody according to claim 2, wherein: the heavy chain nucleotide sequence of the antibody is shown as SEQ ID NO. 11.

7. A full-new sequence anti-VEGF-anti-PD 1 bispecific antibody according to claim 2, wherein: the light chain nucleotide sequence of the antibody is shown as SEQ ID NO. 12.

8. a pharmaceutical composition comprising the antibody of any one of claims 1 ~ 7 and a pharmaceutically acceptable carrier.

9. use of an antibody according ~ any one of claims 1 ~ 7 in the manufacture of a medicament for inhibiting or neutralizing VEGF and PD1 activity.

10. The use according to claim 9, the medicament inhibiting or neutralizing VEGF and PD1 activity for the treatment of cancer.